Stable polymeric micelle-type drug composition and method for the preparation thereof

ABSTRACT

A biocompatible stable composition containing a hydrophobic drug, such as paclitaxel. The composition, which forms a syringeable polymeric micellar solution in aqueous or body fluids, is a freeze-dried product comprising a hydrophobic drug, i.e. paclitaxel, and an amphiphilic block copolymer wherein a hydrophobic group having affinity or attraction with the hydrophobic drug, such as paclitaxel, is incorporated on its end.

This application is based on PCT/KR01/00802, which claims priority,based on a Korean patent application No. 2000-26359 filed May 17, 2000.

TECHNICAL FIELD

The present invention relates to a biocompatible and stable polymericdrug composition capable of forming a micelle in an aqueous environment,said composition comprising an amphiphilic block copolymer of ahydrophilic poly(alkylene glycol) component and a hydrophobicbiodegradable component wherein the hydrophobic biodegradable componentof the copolymer is capped with a modifying group having an affinity orattraction for a hydrophobic drug, and wherein a hydrophobic drug isphysically trapped in the hydrophobic core of the micelle. Thismicelle-forming composition can solubilize the hydrophobic drug in ahydrophilic environment forming a stable hydrophobic drug-containingmicellar solution.

BACKGROUND ART

Many important drugs are hydrophobic and have limited solubility inwater. In order to attain the expected therapeutic effects of suchdrugs, it is usually required that a solubilized form of the drug beadministered to a patient. For this purpose there have been developed anumber of methods which are based on the use of: auxillary solvents;surfactants; soluble forms of the drug, e.g., salts and solvates;chemically modified forms of the drug, e.g., prodrugs; solublepolymer-drug complexes; special drug carriers such as liposomes; andothers. Each of the above methods is hampered by one or more particularproblems, e.g., the method based on the use of a surfactant tosolubilize hydrophobic drugs has problems in that most surfactants arerelatively toxic and precipitation of the hydrophobic drug occurs whensubjected to dilution. European Patent EP 0645145 discloses a method ofsolubilizing a typical poorly water soluble drug, paclitaxel, by use ofCremophor EL™, a polyoxyethylene castor oil derivative. The use of thesesurfactants, however, is restricted due to toxic side effects such ashypersensitivity. They have limitations in that their poor ability tostabilize micelles can cause precipitation of the drug when the micellarsolution is either stored or is to remain in place for an extendedperiod of time.

In recent years, polymeric micelles have been investigated as potentialcarriers for poorly water soluble drugs. Efforts have been made for thepreparation, characterization and pharmaceutical application ofpolymeric micelles. For example, see M. Jones, et al., Polymericmicelles—a new generation of colloidal drug carriers, Eur. J. Pharm.Biopharm. 48(1999) 101–111. Polymeric micelles provide attractivecharacteristics in two major aspects: (a) they can solubilize poorlywater soluble, or hydrophobic drugs in their hydrophobic inner core; and(b) they can avoid uptake of the drug by the RES (reticuloendothelialsystem) or the MPS (mononuclear phagocytes system) in vivo.

Polymeric micelles are characterized by a core-shell structure inaqueous media that results from the amphiphilic block copolymers havinghydrophobic (core) and hydrophilic (shell) segments. A poorly watersoluble drug is entrapped within the hydrophobic core of the micelle.There has been considerable research in the development of A-B, A-H-A,or B-A-B block copolymers having a hydrophilic A block and a hydrophobicB block. As a drug carrier, it is preferred that the hydrophobic B(innermicelle core block) comprises a biodegradable polymer such aspoly-DL-lactide, poly-ε-caprolactone or poly(γ-benzyl-L-aspartate) andthe hydrophilic A (outer micelle shell block) be a polymer such aspolyethylene glycol which is capable of interacting with plasma proteinsand cell membranes.

Polymeric micelles can provide for prolonged systemic circulation timedue to their small size (<100 nm), their hydrophilic shell whichminimizes uptake by the MPS, and their high molecular weight whichprevents renal excretion (K. Katasoka, Design of nanoscopic vehicles fordrug targeting based on micellization of amphiphilic block copolymers,J. Macromol. Sci.—Pure Appl. Chem A31(1994) 1759–1769). Additionally, H.Maeda showed experimental evidence supporting the enhanced permeabilityand retention (EPR) effect of macromolecules in cancer chemotherapy. Thetumor vessels are more leaky and less permiselective than normalvessels, and accumulation of polymeric micelles in tumors is explainedby this increased vascular permeability and the lack of lymphaticdrainage in tumors (H. Maeda, The tumor blood vessel as an ideal targetfor macromolecular anticancer agents, J. Control. Rel. 19(1992)315–324).

Among various pharmaceutical applications of polymeric micelles,research has been focused on the parenteral administration of anticancerdrugs using polymeric micelles because of the above-describedadvantages, such as a long circulation time in vivo, and drug targetingby the EPR effect.

Taxanes, including paclitaxel and its analogues, that exert antitumoractivity due to inhibition of cell proliferation by preventingmicrotuble assembly, are promising anticancer agents and theirpreparation methods and application for chemotherapy have been widelystudied. They are now available from various routes of supply such asextraction from the bark or needles of the pacific yew tree, biologicalmethod of tissue culture, or chemical synthesis. Since paclitaxel ispractically insoluble in water (solubility of less than 0.01 mg/mL),several compositions to solubilize or disperse the drug in infusionfluid have been proposed for parenteral administration to patients.Bristol-Myers Squibb introduced an injectable composition containingpaclitaxel, Taxol®, and this formulation is the only one which has beenapproved for human use by the FDA. Taxol® is a solution in which amixture of paclitaxel and polyethoxylated castor oil (Cremophor® EL,BASF Aktiengesolischaft) is dissolved in alcohol. However, Cremophor® ELhas a potential for inducing various side effects including anaphylacticreactions. Additionally, the Cremophor® EL in the Taxol® formulationcauses the leaking of harmful plasticizers into the infusion fluid fromthe infusion bags or plastic tubes.

Intensive studies have been made in an effort to overcome theshortcomings of the Taxol® formulation, and as a result, severalcompositions containing paclitaxel are known as substitutes for theTaxol® formulation. U.S. Pat. No. 5,877,205 discloses a compositionformulated in such a manner that pacilataxel is dissolved in an organicsolvent followed by addition of secondary solvent to stabilize the drugin solution for subsequent final dilution in an aqueous lipid emulsion.U.S. Pat. No. 5,922,754 discloses another composition comprisingpaclitaxel, an acid, water, and mixture of some organic solvents such astriacetin, alcohol, and Solutol™ (BASF, polyethylene glycol ester of12-hydroxystearic acid).

Although the solution of the above formulation is stable and does notprecipitate for more than 72 hours (3 days) at room temperature whilethe solution of the Taxol® formulation is stable for 27 hours, there isan important limitation to their use in the body because theformulations still contain organic solvents, such as dimethyl acetamide,or excess amounts of Solutol™ (LD₅₀[mouse, iv] of Polyoxyl 20Stearate=0.87 g/kg), which is more toxic than Cremophor EL (LD₅₀[mouse,iv]=2.5 g/kg). [LD₅₀ from Handbook of Pharmaceutical Exipients, 2nd ed.,American Pharmaceutical Association].

Therefore, while polymeric micelles seem to be one of the mostadvantageous carriers for the delivery of poorly water soluble drugs,such as paclitaxel or other anti-cancer agents, problems remain due totheir lack of stability in infusion fluid or body fluid. X. Zhang et al,reported that a diblock copolymer of polylactide andmonomethoxypolyethylene glycol(mPEG) was useful as a carrier ofpaclitaxel (X. Zhang et al. Development of amphiphilic diblockcopolymers as micellar carriers of taxol, Int. J. Pharm. 132(1996)195–206). The formulation dissolves paclitaxel by incorporating the druginto a polymeric micelle in aqueous media. This formulation has anadvantage in that the materials employed in this formulation arenon-toxic and their hydrolysis products are easily eliminated from thebody, thus, overcoming prior art shortcomings in compositions containingpaclitaxel, such as the Taxol® formulation, and formulations shown inU.S. Pat. Nos. 5,877,205 and 5,922,754. The formulation shown in Zhanget al., however, still has a disadvantage in that, due to unstablemicellar formation, the drug is precipitated from the micelle into theaqueous infusion fluid within 48 hours.

Although polymeric micelles would seem to be ideal carriers for poorlywater soluble drugs because of their distinct advantages, such as smallsize, high solubility, simple sterilization, controlled release ofdrugs, the physical stability of such carriers limits their applicationfor pharmaceutical use.

SUMMARY OF THE INVENTION

The present invention provides an improved, stable, hydrophobic drugcontaining polymeric micelle in an aqueous media. The composition of thepresent invention can be stored for longer than three years in asterilized container, without any denaturation of the compounds and thepolymeric micelles formed in the aqueous infusion fluid of the presentinvention are stable for longer than 72 hours (3 days). In addition, theformulation of the present invention causes no side effects to a patientand intravascular administration of the formulation provides improvedbioavailability with high plasma concentration of the drug, e.g.paclitaxel, being achieved.

The present invention provides a stable biodegradable polymericmicelle-type drug composition which comprises: a modified biodegradablepolymeric drug carrier micelle having a hydrophobic drug physicallytrapped within, but not covalently bonded to the drug carrier micelle.The micelle is capable of dissolving in water to form a stable,injectable solution thereof. The drug carrier micelle comprises anamphiphilic block copolymer having a hydrophilic poly(alkylene glycol) Ablock component, and a biodegradable hydrophobic polymer B blockcomponent selected from the group consisting of poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid),poly(ε-caprolactone), and wherein the amphiphilic block copolymer hasterminal ends modified by end groups that have an attraction or affinityfor the hydrophobic drug contained in the micelle core.

The present invention also provides a method for preparing apharmaceutical composition, which comprises the following steps: 1)preparing an amphiphilic block copolymer modified to have end groupingswhich have an affinity or attraction to a hydrophobic drug; 2) preparinga drug-polymer matrix by dissolving a hydrophobic drug and the modifiedblock copolymer in an organic solvent followed by evaporation of thesolvent; 3) preparing an aqueous micellar solution by dissolving thedrug/modified polymer matrix in water; and 4) preparing a finalformulation by freeze-drying the micellar solution followed byappropriate sterilization.

Therefore, the present invention provides for biocompatible, stable,drug containing compositions capable of forming syringeable polymericmicellar solutions in aqueous or body fluids. The composition of thepresent invention is a freeze-dried product comprising a hydrophobicdrug (e.g. paclitaxel) and an amphiphilic block copolymer whereinhydrophobic drug attracting groups are incorporated on its ends. Thecomposition of the present invention also provides i) a shelf life oflonger than three years in a sterilized container, ii) stability oflonger than three days in an infusion fluid, iii) minimal side effectsdue to no use of any toxic excipients or organic solvents, and iv)improved bioavailability indicated by the high concentration of thehydrophobic drug such as paclitaxel achieved in plasma.

Additional features and advantages of the invention will be apparentfrom the detailed description which follows, taken in conjunction withthe accompanying examples, which together illustrate, features of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the NMR spectrum of mPEG—PLA—Bz;

FIG. 2 is the NMR spectrum of mPEG—PLA—Ac; and

FIG. 3 is the NMR spectrum of mPEG—PLA.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made to the exemplary embodiments and specificlanguage will be used herein to describe the same. It will neverthelessbe understood that no limitation of the scope of the invention isthereby intended. Alterations and further modifications of the inventivefeatures illustrated herein, and additional applications of theprinciples of the invention as illustrated herein, which would occur toone skilled in the relevant art and having possession of thisdisclosure, are to be considered within the scope of the invention.

The amphiphilic block copolymer micelle composition of the presentinvention is very effective in solubilizing hydrophobic drugs by way ofphysically incorporating them within the micelle and improving thestability of the drug by means of the affinity or attraction provided bythe end group modifications to the copolymer. The resultingbiodegradable polymeric micelle composition containing the hydrophobicdrug is soluble in water forming a solution that is suitable forsustained-release of the drug in vivo, thereby enhancing the therapeuticeffect of the drug. Such therapeutic effect may be maximized bycontrolling the molecular weights and the relative ratios of thehydrophilic and hydrophobic blocks. Moreover, the composition of thepresent invention can be stored for longer than three years in asterilized container without any denaturation of the components and thepolymeric micelles formed in the aqueous infusion fluid of the presentinvention are stable for longer than 72 hours (3 days). In addition, theformulation of the present invention causes minimal or no side effectsto a patient and intravascular administration of the formulationprovides for improved bioavailability with high plasma concentrations ofthe drug being achieved.

The biodegradable polymeric micelle-type drug composition of the presentinvention, which is capable of forming stable polymeric micelles inaqueous or body fluids, is comprised of a biodegradable modifiedamphiphilic block copolymer having physically entrapped therein one ormore hydrophobic drugs, and when administered, the hydrophobicbiodegradable polymer decomposes in vivo by simple hydrolysis intonon-toxic small molecules.

The modified amphiphilic block copolymer comprises a hydrophilicpoly(alkylene glycol) component and a hydrophobic biodegradable polymercomponent. The polyalkylene glycol suitable for the hydrophiliccomponent in the block copolymer of the present invention is a memberselected from the group consisting of polyethylene glycol, monoalkoxypolyethylene glycol and monoacyloxy polyethylene glycol, wherein themolecular weight of the polyalkylene glycol is preferably within therange of 200˜20,000 Daltons and more preferably, within the range of1,000˜15,000 Daltons.

The hydrophobic biodegradable polymer component of the copolymer of thepresent invention is a member selected from the group consisting ofpolylactides, polycarprolactone, copolymers of lactide and glycolide,copolymers of lactide and carprolactone, copolymers of lactide and1,4-dioxan-2-one, polyorthoesters, polyanhydrides, polyphosphazines,poly(amino acid)s and polycarbonates. Preferably, the hydrophobicbiodegradable polymer component of the copolymer of the presentinvention is a member selected from the group consisting ofpolylactides, polycaprolactone, a copolymer of lactide and glycolide, acopolymer of lactide and caprolactone, and a copolymer of lactide and1,4-dioxan-2-one. The molecular weight of the hydrophobic biodegradablepolymer component is preferably within the range of 500˜20,000 Daltonsand more preferably within the range of 1,000˜10,000 Daltons.

As will be more fully described in connection with Formula I thatfollows, the hydroxy group conventionally found at the end of ahydrophilic polyalkylene glycol can be blocked or capped by a C₁-C₄alkyl group thereby forming an ether capping, such as is found inmonomethoxy polyalkylene glycols (mPEG) or by a C₁-C₄ acyl therebyforming an ester capping, such as is found in monoacyloxy polyalkyleneglycols. The hydroxyl group at the end of a hydrophobic polymer block,such as a polylactide, is capped by acylation thereby forming an estercapping wherein the acyl group contains from 2 to 10 carbon atoms suchas an alkyl, aryl, alkaryl or aralkyl group as will be more fullyexplained. Preferably, the end capping of the hydrophilic block will beby a methoxy group and the end capping of the hydrophobic block will beby an acetyloxy or benzoyloxy group.

The amphiphilic block copolymers can be prepared according to methodsdescribed in U.S. Pat. Nos. 5,683,723 and 5,702,717, hereby fullyincorporated by reference. For example they may be prepared via ringopening bulk polymerization of one of the monomers, such as a lactide,caprolactone, 1,4-dioxan-2-one, or a glycolide, with a polyethyleneglycol derivative in the presence of stannous octoate as a catalyst.Block copolymers having a poly(amino acid) block are prepared by thereaction of an amino acid N-carboxy anhydride with a polyethylene glycolderivative. The hydrophilic polyethylene glycol block is preferably inthe range of 30˜70% by weight of the block copolymer, and mostpreferably 40˜60% by weight.

The improved stability attributable to the present invention is by meansof modifying the block copolymer such that at least one end of the endterminal groups has an affinity or attraction with a hydrophobic drug,which significantly improves the stability of the micelles and the drugsentrapped therein.

Any drug having a water solubility of less than 10 mg/ml can be used asthe “hydrophobic drug” or “poorly water soluble drug” to be incorporatedin the polymeric micelles of the present invention. Examples ofhydrophobic drugs that can be used include anticancer agents,antiinflammatory agents, antifungal agents, antiemetics,antihypertensive agents, sex hormones, and steroids. Typical examples ofthe hydrophobic drugs are anticancer agents such as paclitaxel,taxotane, camptothecin, doxorubicin, daunomycin, cisplatin,5-fluorouracil, mitomycin, methotrexate, and etoposide; antiinflammatoryagents such as indomethacin, ibuprofen, ketoprofen, flubiprofen,dichlofenac, piroxicam, tenoxicam, naproxen, aspirin, and acetaminophen;antifungal agents such as itraconazole, ketoconazole, amphotericin; sexhormones such as testosterone, estrogen, progestone, and estradiol;steroids such as dexamethasone, prednisodone, and triameinolene;antihypertensive agents such as captopril, ramipril, terazosin,minoxidil, and parazosin; antiemetics such as ondanseiron, andgranisetron; antibiotics such as metronidazole, and fusidic acid;cyclosporine; prostagladins; and biphenyl dimethyl dicarboxylic acid.The present invention is particularly useful for administeringanti-cancer drugs such as paclitaxel, taxotane, doxorubicin, cisplatin,carboplatin, 5-FU, etoposide, and camptothecin; sex hormones such astestosterone, estrogen, and estradiol; antifungal agents such asitraconazole, ketoconazole, and amphotericin; steroids such astriamoinolone acetonide, hydrocortisone, dexamethasone, prednisolone,and betamethasone; cyclosporine; and prostagladins. The hydrophobic drugmay be incorporated in the polymeric micelle composition up to 50 wt %based on the total weight of the block copolymer and the drug.

One embodiment of the present invention provides a pharmaceuticalcomposition, which is capable of forming a stable polymeric micelle inaqueous or body fluids. comprising:

-   -   a) a taxane analog; and    -   b) b) a block copolymer which is represented by formula (I)        below:

-   -    wherein R₁ is H, a C₁ to C₄ alkyl, a C₁ to C₄ acyl or

-   -    wherein R₂ is a C₁ to C₉ alkyl, aryl, alkaryl or aralkyl        groups; x is an integer of 20–300, and y is an integer of 15–70.

Representatives of alkyl groups are methyl, ethyl, propyl, and butyl.Representative of an aryl group is phenyl as well as functionallyequivalent heterocyclic groups such as thienyl, furyl, pyridinyl groupsand the like. Representative of an aralkyl grouping is benzyl andrepresentative of an alkaryl group is tolyl. Preferably R₁ is a methylgroup and R₂ is methyl or phenyl group.

The block copolymer of the present invention can be prepared via ringopening bulk polymerization of heterocyclic ester compounds (lactones),such as DL-lactide, glycolide, ε-caprolactone, or p-dioxanone, withpolyethylene glycol or monomethoxy polyethylene glycol in the presenceof stannous octoate. At least one terminal ends of the copolymer iscapped in the manner described with a group such as benzoyl or acetylgroup having affinity or attraction for a hydrophobic drug such aspaclitaxel. One example of the resultant block polymer of this inventionis represented by formula (I). Methods of adding an end group to the endof block copolymer were described in the “Preparation Examples 1a, 1b,and 2”:

[For a benzoyl group]

-   mPEG+DL-lactide→mPEG—PLA—OH (block copolymer having hydroxyl group)-   mPEG—PLA—OH+Cl—(C═O)—C₆H₅ (benzoyl chloride)→-   mPEG—PLA—O—(C═O)—C₆H₅ (block copolymer having a benzoyloxy group)    [For an acetyl group]-   mPEG—PLA—OH+Cl—(C═O)—CH₃ (acetyl chloride)→-   mPEG—PLA—O—(C═O)—CH₃ (block copolymer having an acetyloxy group)

In this case, the block copolymer and the end group are linked by anester bond [—O—(C═O)—], and can be expressed as mPEG—PLA—O—(C═O)—R,where R could be CH₃, C₆H₅, ethyl, propyl, or others.

An alternative method to modify the block copolymer of the presentinvention is by using isocyanate;

-   mPEG—PLA—OH+O—C═N—CH₂CH₃ (ethyl isocyanate)→-   mPEG—PLA—O—(C═O)—NH—CH₂CH₃ (block copolymer having an ethyl    carbamoyloxy group), or-   mPEG—PLA—OH+O—C═N—C₆H₅C(═O)—O—CH₃ (methyl isocyantobenzoate)→-   mPEG—PLA—O—(C═O)—NH—C₆H₅C(═O)—O—CH₃ (block copolymer having a    methoxycarbonyl phenyl carbamoyloxy group)

In this case, the block copolymer and the end group are linked by acarbamate(urethane) bond [—O—(C═O)—N—], and can be expressed asmPEG—PLA—O—(C═O)—NH—R, wherein R is a C₁ to C₉ member selected from thegroup consisting of alkyl, aryl, alkaryl and aralkyl groups.Representatives of alkyl groups are methyl, ethyl, propyl, and butylgroups. Representative of an aryl group is phenyl as well asfunctionally equivalent heterocyclic groups such as thienyl, furyl,pyridinyl, and the like. Representative of an aralkyl group is benzyland representative of an alkaryl group is tolyl. Preferably R₁ is amethyl group and R₂ is a methyl or phenyl group.

Illustratively, the copolymer (10˜200 mg) prepared as described is thendissolved in an organic solvent (1˜5 mL) such as acetonitrile,dichloromethane, or tetrahydrofuran (THF). A poorly water soluble drug(2˜50 mg) such as paclitaxel, is dissolved in the same organic solvent,and then mixed with the polymer solution. A homogeneous drug-polymermatrix is obtained by evaporating the organic solvent at an elevatedtemperature. The drug-polymer matrix is dissolved in water to produce anaqueous micellar solution at a polymer concentration higher than thecritical micelle concentration (CMC). The polymeric micelle having aspherical shape in aqueous media consists of two different regions, ahydrophobic inner core and a hydrophilic outer shell. This particularstructure is due to the amphiphilic properties of the polymer whichconsists of a hydrophobic polylactone block and a hydrophilicpolyethylene glycol block. The hydrophobic drug, such as paclitaxel, istrapped in the inner core of the spherical micelle. The stable micellarcomposition containing paclitaxel, in the hydrophobic core formed by thehydrophobic segments of the copolymer, is prepared by freeze-drying theaqueous micellar solution.

The freeze-dried composition pared by the above-mentioned method can bediluted in an aqueous media, such as 0.9% sodium chloride (normalsaline), 5% dextrose, 5% dextrose and 0.9% sodium chloride, or 5%dextrose in Ringer's solution, to achieve a final paclitaxelconcentration of 0.1˜3.0 mg/mL, more preferably 0.2˜1.5 mg/mL. Thediluted solution is placed in a thermostat at 25° C. At predeterminedtime intervals, 0.5 mL of the solution is taken out with a syringe andfiltered through a 0.45 μm PVDF syringe filter (Milipore, Cat No.SLHV004NL). The drug concentration in the filtered solution is thendetermined by high performance liquid chromatography (HPLC) assay.

Paclitaxel is traditionally administered at a dose of about 175 mg/m².For a human adult with 70 Kg body weight, the surface area and the totalblood volume are about 1.8 m² and 5 L, respectively. When paclitaxel isadministered by one bolus intravenous injection at the indicated dose,initial plasma concentrations of paclitaxel are in the range of0.04˜0.08 mg/mL. Therefore, the stability test for a dilutedconcentration of 0.04˜0.08 g/mL is also carried out at body temperature(37° C.).

The HP1100 series HPLC system (Hewlett-Packard) is used fordetermination of the drug concentration. Peak detection and integrationis performed with HP Chemstation for LC Rev.A.06.01. Chromatographicseparation is achieved with 00G-4012-E0 (Phenomenex) column (250×4.6 mm,5 μm). Paclitaxel and the internal standard were eluted with the mobilephase of actonitrile-water (45:55, v/v) using a flow rate of 1.5 mL/min.Ultraviolet (UV) analysis was performed at a wavelength of 227 nm.Propyl-p-hydroxybenzoate was used for the internal standard.

The terminal end capping groups in the block copolymer play an importantrole in the stability of the hydrophobic drug trapped in the core regionof the micelle formed in aqueous media. The formulations employing thediblock copolymers of polyethylene glycol and polylactone which do nothave the ends capped with groups having an attraction or affinity forthe hydrophobic drug have a drawback in that the drug is precipitatedfrom the micelle into the aqueous infusion fluid within 48 hours due tounstable micellar formation. In order to overcome the precipitation of adrug in the infusion fluid, the block copolymer of the present inventionwas modified terminal hydroxyl groups that are capped with a group whichhas affinity or attraction for the hydrophobic drug. Thus, thehydrophobic drug remains in the hydrophobic core of the micelle for alonger period of time due to the affinity or attraction between the drugand the terminal end capping group of the polymer. As a result, thecomposition provides long-term stability for infusion therapy.Furthermore, the pharmaceutical composition of the present inventionincorporates paclitaxel up to 40% by weight.

Traditionally, prior art formulations are supplied as a concentratedsolution composition in organic solvents, and they are diluted inaqueous media before use. On the contrary, the final formulation of thepresent invention is a freeze-dried composition in a sterilizedcontainer. It is easily dissolved to a concentration of 0.1˜3.0 mg/mL,more preferably 0.2˜1.5 mg/mL, in an appropriate conventional injectionfluid prior to infusion. As the composition contains no solvents and isstored in a very stable freeze-dried solid state, the composition of thepresent invention eliminates any possible denaturation or precipitationof the drug by temperature changes during storage, that is, thecomposition provides a longer shelf life than those in the prior art.

The polymeric micellar solution of the present invention is stable withno precipitation in the infusion fluid for longer than 72 hours (3 days)at room temperature (25° C.). When the composition is diluted to aconcentration of paclitaxel of 0.04˜0.08 mg/mL, i.e. initial plasmaconcentration after one bolus iv injection of the recommended dose ofTaxol® inj., the composition is more stable than the compositionsformulated with the polymers not having the above-described terminal endcapping groups. Furthermore, the composition of the present inventionimproves the paclitaxel plasma concentration in pharmacokineticexperiments with rats, as described below.

The formulation of the present invention does not contain anypotentially harmful material for use in the human body, such as anorganic solvent or Cremophor EL which induce various side effects. Thepolymers incorporated in the composition are biocompatible, they arealready approved for use in the human body from the FDA, and theirhydrolysis products are easily eliminated from the body.

A pharmacokinetic experiment was performed with Sprague-Dawley ratshaving a body weight of 200˜250 g. The freeze-dried compositionformulated by the above-mentioned method was dissolved in normal salineto give a paclitaxel concentration of 1.0 mg/mL and the formulation wasinjected into the tail vein with the does of paclitaxel given being 20mg/kg. At specified time intervals, blood samples were drawn inheparinized tubes from the tail vein. They were centrifuged at 2000 rpmfor 5 minutes for separation. The internal standard, biphenyl dimethyldicarboxylate, was added to the separated plasma for HPLC assay. Thedrug was extracted from the plasma using ethyl acetate, and dried byevaporation of the solvent. The dried product was dissolved inactonitrile-water and the paclitaxel plasma concentration was determinedby HPLC as described above. A standard solution was prepared bydissolving a known amount of paclitaxel in the plasma, acetonitrile, andthe internal standard. The HPLC assay for the stability test wasperformed with the above-described HPLC system. Chromatographicseparation was achieved with a VYDAC (Hesperia) 218MR54 C18 column(250×4.6 mm, 5 μm). Paclitaxel and the internal standard were elutedwith the mobile phase of actonitrile-water, with a linear gradient from30:70 (v/v) to 60:40 (v/v) for 40 minutes, using a flow rate of 1.0mL/min. Ultraviolet (UV) analysis was performed at a wavelength of 227nm. Biphenyl dimethyl dicarboxylate was used for the internal standard.

While the following examples are provided for the purpose ofillustrating certain aspects of the present invention, they are not tobe construed as limiting the scope of the appended claims.

Examples Preparation Example 1a

A diblock copolymer of monomethoxy polyethylene glycol and polylactidehaving a benzoyloxy terminal group. (mPEG—PLA—Bz)

25 grams of monomethoxy polyethylene glycol (mPEG with a molecularweight of 2,000) and DL-lactide which was recrystallized from ethylacetate, and 0.25 g of stannous octoate which was dissolved in 5 mLtoluene, were added to a reactor equipped with a mechanical stirrer anda distillation set. Excess toluene was evaporated at 120° C. Thepolymerization reaction was carried out under vacuum (25 mmHg) for 6hours. The vacuum was released and 50 mL benzoyl chloride was added tocause substitution of the hydrogen atom of the terminal hydroxyl groupby a benzoyl group. The reaction mixture was then agitated for 5 hoursat 100° C. The reaction product was dissolved in chloroform and pouredinto cold diethyl ether (4° C.) to precipitate the polymer. Theprecipitated polymer was washed twice with diethyl ether and dried undervacuum (0.1 mmHg) for 24 hours. The molecular weight of the blockcopolymer (mPEG—PLA—Bz) was determined by nuclear magnetic resonance(NMR) spectroscopy. The NMR spectrum is as shown in FIG. 1.

Preparation Example 1b

A diblock copolymer of monomethoxy polyethylene glycol and polyactidehaving a benzoyloxy terminal group (mPEG—PLA—Bz)

25 grams monomethoxy polyethylene glycol (mPEG with a molecular weightof 2,000) and DL-lactide which was recrystallized from ethyl acetate,and 0.25 g of stannous octoate which was dissolved in toluene (5 mL),were added to a reactor equipped with a mechanical stirrer and adistillation set. Excess toluene was evaporated at 120° C. Thepolymerization reaction was carried out under vacuum (25 mmHg) for 6hours. The reaction product was dissolved in chloroform and poured intocold diethyl ether (4° C.) to precipitate the polymer. The precipitatedpolymer (mPEG—PLA) was washed twice with diethyl ether and dried undervacuum (0.1 mmHg) for 24 hours.

In order to substitute the hydrogen atom of the terminal hydroxyl groupwith a benzoyl group, the above-obtained polymer (mPEG—PLA) (30 g) andbenzoyl chloride (60 mL) were added into a reactor and agitated for 5hours at 100° C. The reaction product was dissolved in chloroform andpoured into cold diethyl ether (4° C.) to precipitate the polymer. Theprecipitated polymer was washed twice with diethyl ether and dried undervacuum (0.1 mmHg) for 24 hours. The molecular weight of the blockcopolymer (mPEG—PLA—Bz) was determined by nuclear magnetic resonance(NMR) spectroscopy. The NMR spectrum is as shown in FIG. 1.

Preparation Example 2

A diblock copolymer of monomethoxy polyethylene glycol and polylactidehaving an acetyloxy terminal group

A diblock copolymer (mPEG—PLA—Ac) was prepared using acetyl chloride (50mL) instead of benzoyl chloride, added to cause substitution of thehydrogen atom of the terminal hydroxyl group by a acetyl group. Themolecular weight was determined by the same procedure described inpreparation Example 1a. The NMR spectrum is as shown in FIG. 2.

Comparative Preparation Example 1

A diblock copolymer of monomethoxy polyethylene glycol and polylactide.

25 grams of monomethoxy polyethylene glycol (mPEG with a molecularweight (mw) of 2,000) and DL-lactide which was recrystallized from ethylacetate, and 0.25 grams of stannous octoate which was dissolved in 5 mLtoluene, were added to a reactor equipped with a mechanical stirrer anda distillation set. Excess toluene was evaporated at 120° C. Thepolymerization reaction was carried out under vacuum (25 mmHg) for 6hours. The reaction product was dissolved in chloroform and poured intocold diethyl ether (4° C.) to precipitate the polymer. The precipitatedpolymer was washed twice with diethyl ether and dried under vacuum (0.1mmHg) for 24 hours. The molecular weight of the block copolymer(mPEG—PLA) was determined by nuclear magnetic resonance (NMR)spectroscopy. The NMR spectrum is as shown in FIG. 3.

Examples 1a˜2 Stability of the Composition in Infusion Fluid

The polymers (190 mg) prepared in Examples 1a, 1b, and 2, were dissolvedin acetonitrile (2 mL). Paclitaxel (10 mg), which was dissolved inacetonitrile (1 mL), was mixed with the polymer solution. A homogeneousdrug-polymer matrix was obtained by evaporating the organic solvent at60° C. under nitrogen flow followed by vacuum (0.1 mmHg) drying for 24hours. The aqueous micellar solution was prepared by dissolving thedrug-polymer matrix in distilled water (2 mL). The solution was thenfreeze-dried at −50° C. for 24 hours.

In order to dilute the formulation to a concentration for infusion(paclitaxel concentration of 1.0 mg/mL), the freeze-dried composition(100 mg), prepared as described above, and saline (5 mL) were added to avial and mixed with a Vortex Mixer. This diluted solution was thenplaced in a thermostat at 25° C. At given time intervals, a 0.2 mLsolution was removed by a syringe and filtered through a 0.45 μm PVDFsyringe filter (Milipore, Cat No. SLHV004NL). The drug concentration inthe solution was then determined by HPLC assay as described above. Theresults are shown in Table 1.

Comparative Example 1

The freeze-dried compositions and micellar solutions were prepared bythe same procedure as described in Example 1, using the polymersprepared in comparative Example 1. The results of the stability test areshown in Table 1.

Comparative Example 2

(Taxol® Formulation)

Taxol® (Britol-Myers Squibb) formulation was diluted to a concentrationsuitable for infusion (paclitaxel concentration of 1.0 mg/mL) in normalsaline, and the stability test was carried out by the same procedure asdescribed in Example 1. The results are shown in Table 1.

TABLE 1 Stability of the Composition in Infusion Fluid (1.0 mg/mL) at25° C. Remained Drug (%) No. Polymer 0 hr 24 hr 48 hr 72 hr Example 1amPEG-PLA-Bz 100 100 99.3 98.7 1b mPEG-PLA-Bz 100 100 99.5 98.7 2mPEG-PLA-Ac 100 99.5 98.7 97.5 Comparison 1 mPEG-PLA 100 98.0 75.3 62.42 Cremophor EL^(a)) 100 95.0 82.7 67.0 ^(a))Test was carried out usingTaxol ® (Britol-Myers Squibb) formulation.

As shown in Table 1, when the paclitaxel was incorporated in thecomposition employing a polymer with a functional group at its endhaving chemical attraction to it, more than 90% of the drug remainedincorporated in the polymeric micelles at a concentration in theinfusion fluid of (1.0 mg/mL) for 3 days at 25° C., while less than 70%of the drug remained in the case of the Taxol® (Britol-Myers Squibb)formulation containing Cremophor EL or the compositions not employingthe functional groups.

Examples 3˜4 Stability of the Composition at a Plasma Concentration

0.5 mL of the aqueous micellar solutions prepared in Example 1a and 2were diluted with normal saline (12.5 mL) to give a paclitaxelconcentration of 0.04 mg/mL, which is below the plasma concentrationwhen administered by one bolus iv injection at the normal dose ofpaclitaxel (175 mg/m²). This diluted solution was then placed in athermostat at 37° C. At given time intervals, a 0.5 mL sample wasremoved by a syringe, and filtered through a 0.45 μm PVDF syringe filter(Milipore, Cat No. SLHV004NL). The drug concentration in the solutionwas then determined by HPLC assay as described above. The results areshown in Table 2.

Comparative Example 3

The stability test was carried out by the same procedure as described inExample 3, using the aqueous micellar solution prepared in ComparativeExample 1. The results are shown in Table 2.

Comparative Example 4

(Taxol® Formulation)

A Taxol® (Britol-Myers Squibb) formulation was diluted in normal salineto a concentration of 0.04 mg/mL, and the stability test was carried outby the same procedure as described in Example 3. The results are shownin Table 2.

TABLE 2 Stability at a plasma concentration (paclitaxel 0.04 mg/mL), 37°C. Remained Drug (%) No. Polymer 0 hr 6 hr 12 hr 24 hr 48 hr 72 hr Exa-3 mPEG-PLA-Bz 100 100 100 100 100 95.6 mple 4 mPEG-PLA-Ac 100 100 100100 100 94.2 Com- 3 mPEG-PLA 100 91.6 54.5 36.8 29.1 25.6 parison 4CremophorEL^(a)) 100 90.3 58.0 43.5 31.8 27.7 ^(a))Test was carried outusing Taxol ® (Britol-Myers Squibb) formulation.

As shown in Table 2, the formulation of the present invention exhibitedimproved stability at a concentration below the initial drug plasmaconcentration corresponding to one bolus iv injection of a normal doseof paclitaxel (175 mg/m²).

Examples 5˜6 Paclitaxel Plasma Concentration in Rat

Paclitaxel compositions for injection were prepared by dissolving thefreeze-dried compositions prepared in Examples 1a and 2 in normal salineto give a concentration of 1.0 mg/mL. According to the proceduredescribed in the pharmacokinetic experiment, the compositions wereinjected into the tail vein of Sprague-Dawley rats having body weightsof 200˜250 g, with the dose of paclitaxel being 20 mg/kg. At given lineintervals, blood samples were drawn in heparinized tubes from the tailvein. The drug plasma concentration was determined by HPLC according tothe above-described procedure and the results are shown in Table 3.

Comparative Example 5

The pharmacokinetic experiments were carried out by the same procedureas described in Example 5, using the aqueous micellar solution preparedin the Comparative Example 1. The results are shown in Table 3.

Comparative Example 6

(Taxol® formulation)

A Taxol® (Britol-Myers Squibb) formulation was diluted to aconcentration of 1.0 mg/mL in normal saline, and the pharmacokineticexperiment was carried out by the same procedure described in Example 5.The results are shown in Table 3.

TABLE 3 Paclitaxel Plasma Concentration in Rat Paclitaxel PlasmaConcentration (μg/mL) 30 120 240 360 No. Polymer 3 min min min min minExample 5 mPEG-PLA- 212.2 61.9 20.7 6.1 2.4 Bz 6 mPEG-PLA- 175.8 47.314.5 5.0 2.1 Ac Comparison 5 mPEG-PLA 40.6 23.4 7.4 2.2 0.1 6 Cremophor105.5 43.0 13.8 5.3 2.1 EL^(a)) ^(a))Test was carried out using Taxol ®(Britol-Myers Squibb) formulation.

As shown in Table 3, the formulation of the present invention exhibited,in rats, superior drug plasma concentrations compared to the Taxol®formulation or the compositions not employing hydrophobic groups at theends of polymer. In other words, the formulation of the presentinvention provides for improved bioavailability of paclitaxel whenadministered by intravenous infusion.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims. It is to be understood that the above examples areillustrative of application of the principles of the present invention.Numerous modifications and alternative arrangements can be devisedwithout departing from the spirit and scope of the present invention.The present invention has been described above in connection with theexemplary embodiments(s) of the invention. It will be apparent to thoseof ordinary skill in the art that numerous modifications can be madewithout departing from the principles and concepts of the invention asset forth in the claims.

1. A composition capable of forming a polymeric micelle in a body fluid or an aqueous medium, said composition comprising an amphiphilic block copolymer having a hydrophilic A block component and a hydrophobic biodegradable B block component, wherein the hydrophobic biodegradable B block component of the copolymer is capped with an acyl group or carbamyl group of C₁ to C₉ alkyl, aryl, alkaryl or aralkyl group.
 2. The composition of claim 1, wherein the amphiphilic block copolymer is selected from the group consisting of AB diblock and BAB triblock copolymers.
 3. The composition of claim 1, wherein the amphiphilic block copolymer is represented by formula (I) below:

wherein x is an integer of 20–300, and y is an integer of 15–70, R₁ is H, a C₁ to C₄ alkyl, a C₁ to C₄ acyl or is represented by formula (II):

wherein R₂ is a C₁ to C₉ alkyl, aryl, alkaryl or aralkyl group, or NH—R₃ wherein R₃ is a C₁ to C₉ alkyl, aryl, alkaryl or aralkyl group, x is an integer of 20–300, and y is an integer of 15–70.
 4. The composition of claim 3, wherein R₂ is a member selected from the group consisting of methyl, ethyl, propyl and butyl.
 5. The composition of claim 3, wherein R2 is a phenyl group or a heterocyclic group selected from the group consisting of thienyl, furyl, pyridinyl groups.
 6. The composition of claim 3, wherein R2 is a benzyl group.
 7. The composition of claim 3, wherein R2 is a tolyl group.
 8. The composition of claim 3, wherein R1 is a methyl group and R2 is a methyl, phenyl or enthylamino group.
 9. The composition of claim 2, wherein the hydrophilic A block component is within a range of 40 to 80 wt % based on total weight of the block copolymer.
 10. The composition of claim 2, wherein the hydrophilic A block component is poly(ethylene glycol) or monomethoxy poly(ethylene glycol).
 11. The composition of claim 1, wherein the block copolymer has an average molecular weight of from 1,000 to 15,000 Daltons.
 12. The composition of claim 1, wherein the hydrophobic biodegradable polymer B block component is selected from the group consisting of a polylactide, copolymer of lactide and glycolide, a copolymer of caprolactone and glycolide, polycaprolactone, a polyanhydride, a polyorthoester, a copolymer of lactide and 1,4-dioxan-2-one, and a copolymer of caprolactone and 1,4-dioxan-2-one.
 13. A hydrophobic drug containing polymeric composition capable of forming stable polymeric micelles in an aqueous environment, said composition comprising a hydrophobic drug and a composition of claim 1, wherein in an aqueous environment said drug is physically entrapped within, but not covalently bound to, a hydrophobic core formed by the hydrophobic B block component and its terminal hydrophobic group.
 14. The composition of claim 13, wherein the content of the hydrophobic drug is up to 50 wt % based on the total weight of the block copolymer and the drug.
 15. The composition of claim 13, wherein the hydrophobic drug has a solubility of less than 10 mg/mL.
 16. The composition of claim 13, wherein the hydrophobic drug is selected from the group consisting of anticancer agents, antifungal agents, steroids, antiinflammatory agents, sex hormones, immunosuppressants, antiviral agents, anesthetics, antiemetics, and antihistamine agents.
 17. The composition of claim 13, wherein the hydrophobic drug is selected from the group consisting of a taxane analog, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, cyclosporine A, amphotericin B, itraconazole, ketoconazole, indomethacin, testosterone, estradiol, dexamethasone, prednisolone, and triamcinolone acetonide.
 18. The composition of claim 17, wherein the hydrophobic drug is a taxane analog.
 19. The composition of claim 18, wherein the taxane analog is paclitaxel.
 20. An aqueous formulation for parenteral administration of a taxane analog comprising the composition according to claim 18, which is dissolved in an aqueous medium and has a concentration of the taxane analog in the range of 0.1˜3 mg/mL.
 21. The aqueous formulation of claim 20, wherein the aqueous medium is a 0.9% sodium chloride water solution, 5% dextrose water solution, 5% dextrose and 0.9% sodium chloride water solution, or a 5% dextrose Ringer's solution.
 22. A method of preparing the composition according to claim 13, which is capable of forming a stable polymeric micelle in aqueous environments, comprising the steps of: a) preparing a drug-polymer mixture by dissolving the amphiphilic block copolymer of claim 1 and a hydrophobic drug in an organic solvent followed by evaporation of the solvent; b) dissolving the drug-polymer mixture in an aqueous environment to obtain a stable micellar solution; and, c) freeze-drying the aqueous micellar solution.
 23. The method of claim 22, wherein the content of the hydrophobic drug is up to 50 wt % based on the total weight of the block copolymer and the drug.
 24. The method of claim 22, wherein the hydrophobic drug has a solubility of less than 10 mg/mL.
 25. The method of claim 24, wherein the hydrophobic drug is selected from the group consisting of a taxane analog, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, cyclosporine A, amphotericin B, itraconazole, ketoconazole, indomethacin, testosterone, estradiol, dexamethasone, prednisolone, and triamcinolone acetonide.
 26. The method of claim 25, wherein the hydrophobic drug is a taxane analog.
 27. The method of claim 26, wherein the taxane analog is paclitaxel.
 28. A hydrophobic drug containing polymeric composition capable of forming stable polymeric micelles in an aqueous environment, said composition comprising a hydrophobic drug and a composition of claim 3, wherein said drug is physically entrapped within, but not covalently bound to, a hydrophobic core formed by the hydrophobic B block component and its terminal hydrophobic group.
 29. The composition of claim 28, wherein the content of the hydrophobic drug is up to 50 wt % based on the total weight of the block copolymer and the drug.
 30. The composition of claim 28, wherein the hydrophobic drug has a solubility of less than 10 mg/mL.
 31. The composition of claim 28, wherein the hydrophobic drug is selected from the group consisting of anticancer agents, antifungal agents, steroids, antiinflammatory agents, sex hormones, immunosuppressants, antiviral agents, anesthetics, antiemetics, and antihistamine agents.
 32. The composition of claim 28, wherein the hydrophobic drug is selected from the group consisting of a taxane analog, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, cyclosporine A, amphotericin B, itraconazole, ketoconazole, indomethacin, testosterone, estradiol, dexamethasone, prednisolone, and triamcinolone acetonide.
 33. The composition of claim 32, wherein the hydrophobic drug is a taxane analog.
 34. The composition of claim 33, wherein the taxane analog is paclitaxel.
 35. An aqueous formulation for parenteral administration of a taxane analog comprising the composition according to claim 33, which is dissolved in an aqueous medium and has a concentration of the taxane analog in the range of 0.1˜3 mg/mL.
 36. The aqueous formulation of claim 35, wherein the aqueous medium is a 0.9% sodium chloride water solution, 5% dextrose water solution, 5% dextrose and 0.9% sodium chloride water solution, or a 5% dextrose in Ringer's solution.
 37. A method of preparing the composition according to claim 28, which is capable of forming a stable polymeric micelle in aqueous environments, comprising the steps of: a) preparing a drug-polymer mixture by dissolving the amphiphilic block copolymer of claim 3 and a hydrophobic drug in an organic solvent followed by evaporation of the solvent; b) dissolving the drug-polymer mixture in an aqueous environment to obtain a stable micellar solution; and, c) freeze-drying the aqueous micellar solution.
 38. The method of claim 37, wherein the content of the hydrophobic drug is up to 50 wt % based on the total weight of the block copolymer and the drug.
 39. The method of claim 37, wherein the hydrophobic drug has a solubility of less than 10 mg/mL.
 40. The method of claim 39, wherein the hydrophobic drug is selected from the group consisting of a taxane analog, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, cyclosporine A, amphotericin B, itraconazole, ketoconazole, indomethacin, testosterone, estradiol, dexamethasone, prednisolone, and triamcinolone acetonide.
 41. The method of claim 37, wherein the hydrophobic drug is a taxane analog.
 42. The method of claim 38, wherein the taxane analog is paclitaxel. 